Increased Angiogenesis by Exosomes Secreted by Adipose-Derived Stem Cells upon Lipopolysaccharide Stimulation

Exosomes secreted by adipose-derived stem cells (ADSCs) enhance angiogenesis and wound healing. However, in clinical settings, wounds may be infected by various bacteria or pathogens. We investigated whether human ADSCs stimulated with lipopolysaccharide (LPS) secrete exosomes (ADSC-LPS-exo) that augment the angiogenesis of human umbilical vein endothelial cells (HUVECs). ExoQuick-TC exosome precipitation solution was used to purify exosomes from human ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h. The uptake of ADSC-LPS-exo significantly induced the activation of cAMP response element binding protein (CREB), activating protein 1 (AP-1), and nuclear factor-κB (NF-κB) signaling pathways and increased the migration of and tube formation in HUVECs. RNA interference with CREB, AP-1, or NF-κB1 significantly reduced the migration of and tube formation in HUVECs treated with ADSC-LPS-exo. An experiment with an antibody array for 25 angiogenesis-related proteins revealed that only interleukin-8 expression was significantly upregulated in HUVECs treated with ADSC-LPS-exo. In addition, proteomic analysis revealed that eukaryotic translation initiation factor 4E, amyloid beta A4 protein, integrin beta-1, and ras-related C3 botulinum toxin substrate 1 may be potential candidates involved in ADSC-LPS-exo-mediated enhanced angiogenesis.


Introduction
Cell-based therapies, such as those comprising adipose-derived stem cells (ADSCs), are considered promising for improving wound healing [1], even in difficult situations such

Uptake of ADSC-exo into HUVECs
The uptakes of labeled exosomes by the cultured HUVECs were found followin incubation of the exosomes with HUVECs for 24 h (Figure 2). The green fluorescen CFSE and red fluorescence of AO found inside the cells revealed the uptake of p and RNAs, respectively, of the ADSC-exo into the HUVECs.

Uptake of ADSC-exo into HUVECs
The uptakes of labeled exosomes by the cultured HUVECs were found following the incubation of the exosomes with HUVECs for 24 h (Figure 2). The green fluorescence of CFSE and red fluorescence of AO found inside the cells revealed the uptake of protein and RNAs, respectively, of the ADSC-exo into the HUVECs.

Induction of Signal Transduction Pathways after ADSC-LPS-exo Treatment
As shown in Figure 3, the experiments with Cignal Finder Reporter Arrays in HUVECs, which could detect 45 signal transduction pathways encoded by firefly luciferase reporter genes, revealed that the uptake of ADSC-LPS-exo significantly induced the activation of CREB, AP-1, and NF-κB signal transduction pathways in cells transfected with ADSC-exo. The expression of CREB, AP-1, and NF-κB1 genes in HUVECs following ADSC-LPS-exo treatment was effectively knocked down by the transfection of pooled siRNAs for CREB, AP-1, and NF-κB1 ( Figure 4).

Uptake of ADSC-exo into HUVECs
The uptakes of labeled exosomes by the cultured HUVECs were found following the incubation of the exosomes with HUVECs for 24 h (Figure 2). The green fluorescence of CFSE and red fluorescence of AO found inside the cells revealed the uptake of protein and RNAs, respectively, of the ADSC-exo into the HUVECs.  indicated the uptake of protein and RNAs, respectively, by the ADSC-exo into the HUVECs. DAPI: 4′,6-diamidino-2-phenylindole; CFSE: carboxyfluorescein succinimidyl diacetate ester; AO: acridine orange.

Induction of Signal Transduction Pathways after ADSC-LPS-exo Treatment
As shown in Figure 3, the experiments with Cignal Finder Reporter Arrays in HU-VECs, which could detect 45 signal transduction pathways encoded by firefly luciferase reporter genes, revealed that the uptake of ADSC-LPS-exo significantly induced the activation of CREB, AP-1, and NF-κB signal transduction pathways in cells transfected with ADSC-exo. The expression of CREB, AP-1, and NF-κB1 genes in HUVECs following ADSC-LPS-exo treatment was effectively knocked down by the transfection of pooled siR-NAs for CREB, AP-1, and NF-κB1 ( Figure 4). . Activated signal transduction pathways in Cignal Finder Reporter Arrays at 24 h after the uptake of ADSC-LPSexo into HUVECs, compared to those treated with ADSC-exo. The reporter array had 45 inducible transcription factorresponsive constructs, which controlled firefly luciferase reporter gene expression, in duplicate, in a 96-well plate. The Renilla luciferase gene was included in the wells for the normalization of transfection efficiency. (*, a p-value < 0.05 with at least 5-fold expression of the luminescence). . RT-qPCR was performed to validate the effective knockdown of the CREB, AP-1 and NF-κB1 genes of HUVECs by the transfection of pooled siRNAs for CREB, AP-1, and NF-κB1 from 10 nM to 40 nM, followed by 30 µ g ADSC-LPS-exo treatment for 24 h. The HUVECS treated with ADSC-LPS-exo alone were used as controls. The HUVECs treated with scramble siRNA first, followed by ADSC-LPS-exo treatment were used as mock control. n = 6 for each condition. (*, a p-value < 0.05 with at least 2-fold reduced expression of the gene). . Activated signal transduction pathways in Cignal Finder Reporter Arrays at 24 h after the uptake of ADSC-LPS-exo into HUVECs, compared to those treated with ADSC-exo. The reporter array had 45 inducible transcription factor-responsive constructs, which controlled firefly luciferase reporter gene expression, in duplicate, in a 96-well plate. The Renilla luciferase gene was included in the wells for the normalization of transfection efficiency. (*, a p-value < 0.05 with at least 5-fold expression of the luminescence).

Angiogenesis after ADSC-LPS-exo Treatment
In comparison with the HUVECs transfected with scramble siRNAs and treated with ADSC-exo, the cell migration and tube formation of HUVECs transfected with 10 nM siRNA-CREB, siRNA-AP-1, siRNA-NF-κB1, or scramble siRNAs, followed by ADSC-LPSexo treatment, were measured. The results revealed that HUVECs treated with ADSC-LPS-exo had significantly enhanced cell migration ( Figure 5) and tube formation ( Figure 6), which manifested as increased total vessel lengths and the total number of junctions detected by Angiotool, compared to those treated with ADSC-exo. In addition, transfection with siRNA-CREB, siRNA-AP-1, and siRNA-NF-κB1 significantly decreased the enhanced cell migration and tube formation following ADSC-LPS-exo treatment. . Activated signal transduction pathways in Cignal Finder Reporter Arrays at 24 h after the uptake of ADSC-LPSexo into HUVECs, compared to those treated with ADSC-exo. The reporter array had 45 inducible transcription factorresponsive constructs, which controlled firefly luciferase reporter gene expression, in duplicate, in a 96-well plate. The Renilla luciferase gene was included in the wells for the normalization of transfection efficiency. (*, a p-value < 0.05 with at least 5-fold expression of the luminescence).

Angiogenesis after ADSC-LPS-exo Treatment
In comparison with the HUVECs transfected with scramble siRNAs and treated with ADSC-exo, the cell migration and tube formation of HUVECs transfected with 10 nM siRNA-CREB, siRNA-AP-1, siRNA-NF-κB1, or scramble siRNAs, followed by ADSC-LPSexo treatment, were measured. The results revealed that HUVECs treated with ADSC-LPS-exo had significantly enhanced cell migration ( Figure 5) and tube formation ( Figure  6), which manifested as increased total vessel lengths and the total number of junctions detected by Angiotool, compared to those treated with ADSC-exo. In addition, transfection with siRNA-CREB, siRNA-AP-1, and siRNA-NF-κB1 significantly decreased the enhanced cell migration and tube formation following ADSC-LPS-exo treatment.

Expression of Angiogenesis-Related Proteins
The Proteome Profiler Human Angiogenesis Antibody Array was used to measure the expression of angiogenesis-related proteins in HUVECs following treatment with 30 µ g ADSC-LPS-exo against those treated with ADSC-exo at the same dosage. Of the measured 25 human angiogenesis-related proteins, only interleukin-8 (IL-8) was significantly upregulated (Figure 7), by around threefold in HUVECs following treatment with ADSC-LPS-exo compared to ADSC-exo. There were no significant changes in the other 52 angiogenesis-related proteins between HUVECs treated with ADSC-LPS-exo and ADSC-exo.

Expression of Angiogenesis-Related Proteins
The Proteome Profiler Human Angiogenesis Antibody Array was used to measure the expression of angiogenesis-related proteins in HUVECs following treatment with 30 µg ADSC-LPS-exo against those treated with ADSC-exo at the same dosage. Of the measured 25 human angiogenesis-related proteins, only interleukin-8 (IL-8) was significantly upregulated (Figure 7), by around threefold in HUVECs following treatment with ADSC-LPS-exo compared to ADSC-exo. There were no significant changes in the other 52 angiogenesisrelated proteins between HUVECs treated with ADSC-LPS-exo and ADSC-exo. Relative expression of angiogenesis-related proteins in the cell lysates of HUVECs following treatment with 30 µ g ADSC-LPS-exo or ADSC-exo was detected in quadruplicates using The Proteome Profiler Human Angiogenesis Antibody Array, which can detect 25 human angiogenesis-related proteins simultaneously with chemiluminescent detection reagents (*, a p-value < 0.05 with at least a 2-fold difference).

Discussion
Many studies have demonstrated that ADSCs can enhance wound healing in chronic wounds [32][33][34] or diabetic wounds [2,35]. Our findings revealed that pretreatment of AD-SCs with LPS can produce exosomes carrying potent molecules for enhanced angiogenesis. This phenomenon is related to the activation of CREB, AP-1, and NF-κB signal transduction pathways and IL-8 production in recipient HUVECs during exosome treatment. Proteomic analysis of ADSC-LPS-exo revealed the notable presence of EIF4E, APP, ITGB1, and RAC1, which may be potential candidates involved in exosome-mediated enhanced angiogenesis.
NF-κB, AP-1, and CREB are known to play a pivotal role in angiogenesis because multiple transcription factor-binding sites for NF-κB, AP-1, CREB, and hypoxia-inducible factor (HIF) have been identified within the vascular endothelial growth factor (VEGF) promoter [36]. Although NF-κB and AP-1 are at the receiving end of different signaling pathways, they are often activated by the same stimuli and simultaneously regulate common target genes implicated in angiogenesis [37][38][39]. For example, NF-κB can mediate VEGF regulation via the AP-1 subunit c-Fos [40] and another AP-1 sub-unit, JunB [37]. The cAMP pathway is known to stabilize endothelial barrier function and maintain vascular physiology. CREB activation, with CREB binding to the VEGF promoter region, is essential for VEGF expression [41][42][43][44][45]. Promoter analysis revealed that the deletion of the CREB site in the proximal region of the promoter markedly reduced VEGF-induced promoter activity, whereas deletion of the upstream NF-κB site had a moderate effect [46]. In addition to angiogenesis [47], CREB is involved in multiple signaling pathways that regulate cell differentiation, proliferation, and migration [48].
IL-8 is a pro-inflammatory chemokine that belongs to the CXC sub-family and has been shown to enhance angiogenesis, increase proliferation and survival, and promote the migration of endothelial cells [49][50][51]. In addition, it correlates with angiogenesis in in vivo models [52,53]. The human IL-8 gene is transcriptionally regulated by NF-κB, AP-1, CREB, CAAT/enhancer-binding protein β (C/EBPβ, also known as NF-IL-6), and C/EBP homologous protein (CHOP) [54]. IL-8 expression is primarily regulated by NF-κB-and/or AP-1-mediated transcriptional activity [52,55]. In Trichomonas vaginalis infection, NF-κB and CREB are involved in IL-8 production in human neutrophils [56]. Human neutrophils may promote angiogenesis via a paracrine feedforward mechanism involving endothelial IL-8 [51]. The biological effects of IL-8 are mediated through the binding of IL-8 to two cellsurface G-protein-coupled receptors, termed as CXCR1 and CXCR2 [49,57,58], with a high affinity [59]. It has been reported that HUVECs constitutively express CXCR1 and CXCR2 mRNA and proteins. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube formation, whereas the neutralization of IL-8 by anti-IL-8 antibody blocked IL-8-mediated capillary tube formation [50]. The mechanism of IL-8 angiogenesis regulation may be due to the enhanced proliferation and survival of endothelial cells by the differential expression of anti-apoptotic genes and, in part, by the activation of MMP-2 and MMP-9 [50].
Proteomic analysis revealed that EIF4E, APP, ITGB1, and RAC1 may be potential candidates for ADSC-LPS-exo-mediated enhanced angiogenesis. In eukaryotes, most mRNAs are translated in a cap-dependent manner. The mRNA 5 cap-binding protein EIF4E is a key player in controlling mRNA translation, a critical process in regulating cell growth, proliferation, and differentiation. Overexpression of EIF4E results in a dramatic increase in VEGF and fibroblast growth factor-2 (FGF-2), two potent angiogenic agents [60,61]. In contrast, blocking EIF4E signaling selectively inhibited angiogenesis in human endothelial cells in vitro [62] and in vivo [63,64].
APP has been reported to be highly expressed in the endothelium of neoforming vessels [65]. The proteolytic cleavage product of APP by β-and γ-secretases mediates sprouting angiogenesis and the formation of new blood vessels [66]. In addition, inhibitors of β-and γ-secretases would inhibit angiogenesis [65].
ITGB1 is a membrane-anchored subunit of many integrins that serve as mechanosensory proteins in endothelial cells [67,68]. Integrins are heterodimeric transmembrane re-ceptors that mediate cell-cell interactions and crosstalk between cells and the extracellular matrix [69]. ITGB1 is essential for blood vessel formation during embryonic development, postnatal vascular remodeling, and vessel maturation [70][71][72]. Loss-and gain-of-function studies have shown that endothelial ITGB1 is involved in angiogenesis and vessel wall remodeling [70,71]. Antibody blockades demonstrate that ITGB1 is functionally important for the migration of endothelial cells [73].
RAC1 is a GTPase that belongs to the RAS superfamily of small GTP-binding proteins (Rho GTPases), which act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements [74]. In endothelial cells, RAC1 controls cell migration and cell-cell junctions, thereby regulating the permeability and formation of vessels [75]. A study revealed that IL-8-upregulated RAC1 increased the migration of HUVECs [76]. In the early stage of metastasis, oncogenic cells undergoing epithelial-mesenchymal transition can communicate with endothelial cells via exosomal Rac1/PAK2 as angiogenic promoters [77].
Although recent reports have implicated exosomes to act in intercellular signaling, their effect in modulating signaling pathways in recipient cells is far from being completely elucidated. Outlining these complex networks may expand our knowledge of the underlying mechanisms involved in the function of exosomes under different stimuli for intercellular communication. However, the current understanding of these "discarded cargoes" is quite limited. EV cargo composition is complex and consists of hundreds to thousands of different proteins, unique lipids, some DNA and mRNA, microRNA, small nucleolar RNA, mitochondrial RNA, and long non-coding RNA (lncRNA) [78]. Although it has been implied that exosomal cargo appears to act in a combinatorial manner when communicating with the recipient cells [79], the molecular cargo mediates specific functions of the exosomes and they do so singly or in combination with other exosomal cargoes, which are still unknown. In addition, whether all exosome cargoes are selectively sorted upon various stimuli to act in different environments remains to be determined.
Furthermore, other limitations of this study should be acknowledged. First, 168 abundant exosomal proteins with 10 hub proteins were identified in ADSC-LPS-exo. The four proteins that are suggested to explain the function of angiogenesis were based on a literature search. It cannot be excluded that angiogenesis may be induced partly or synergistically by other exosomal proteins. In addition, Western blot analyses of each protein or phosphorylated protein with investigation of the function of these proteins on endothelial cells is necessary to confirm the conclusions. Moreover, the effect on angiogenesis by exosomes secreted from ADSCs upon LPS stimulation may rely on factors other than the protein cargo inside exosomes, such as microRNAs [80,81] or lncRNAs [82], which are known to mediate the function of ADSCs on angiogenesis, and should be considered accordingly. Furthermore, LPS, the stimulator used in this study, is only one of a common bacterial toxin component in infectious wounds. This study only investigated one endotoxin found in a complex bacterial wound. Thus, it is considerably different from the milieu that mimics a bacterial wound. There are many other bacterial or fungal components, such as lipoproteins, lipoteichoic acid (LTA; Gram-positive bacteria only), lipoarabinomannan (mycobacteria only), zymosan (yeast), single-or double-stranded RNAs, and flagellin, whose modulatory effects on exosomes secreted by ADSCs remain unexplored. As such, in vivo studies may be necessary to provide valuable information regarding the function of ADSC-exo in complex wounds.

Exosome Isolation
The exosomes were purified from the ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h using ExoQuick-TC TM exosome precipitation solution (EXOTC50A-1, System Biosciences, Palo Alto, CA, USA). The media were centrifuged at 3000× g for 15 min, and the supernatant was transferred into a new tube, followed by the addition of equal volumes of the ExoQuick-TC TM solution. After mixing, supernatants were refrigerated at 4 • C overnight for at least 12 h and then centrifuged at 1500× g for 30 min. The supernatant was discarded, and the pellet was resuspended in PBS and used for further experiments.

Characterization of Exosomes
Under the guidelines of the Minimal Information for Studies of Extracellular Vesicles (MISEV2018) [83], characterization of isolated exosomes was performed to validate the expression of positive and negative exosomal surface markers, detect the morphology and bi-lipid layer structure, and measure the diameters and size distribution of the exosomes. With the culture medium used as a control, Western blotting for the expression of exosomal surface markers of the isolated exosomes was performed in triplicates. The total protein of exosomes was separated by polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). To block the membrane, 5% skim milk in PBS/Tween-20 membranes was used, followed by the subsequent incubation of primary antibodies against four positive proteins for the exosomes, including CD9 (cat. #ab92726, 1:1000; Abcam, Cambridge, MA, USA), CD81 (cat. #ab109201, 1:1000; Abcam), flotillin-1 (cat. #18634, 1:1000; Cell Signaling Technology, Danvers, MA, USA), TSG101 (cat. #ab30871, 1:1000; Abcam). The primary antibody against calnexin (cat. #ab22595; 1:1000; Abcam) was used as a negative control protein, at 4 • C. Membranes were then washed with 0.1% TBS/Tween 20 for 10 min, three times. The incubation was performed at 37 • C with horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. #NA931; GE Healthcare Amersham, Piscataway, NJ, USA) for 2 h. A FluorChem SP imaging system (Alpha Innotech, San Leandro, CA, USA) was used to detect and quantify the protein expression.
For the TEM analyses, we fixed 10 µL exosomes with 2.5% glutaraldehyde for 2 h and then added these exosomes to a 200 mesh Formvar with carbon stabilization. A transmission electron microscope HT-7700 (Hitachi, Tokyo, Japan) at 100 kV was used to analyze the exosome samples which were stained on grids with 2% uranyl acetate for 1 h.
The particle diameters of the isolated exosomes were measured using a Zetasizer Nano-ZS DLS system (Malvern, Montréal, QC, Canada) in triplicates. Briefly, each sample was loaded into an ultraviolet microcuvette (BRAND; Essex, CT, USA) in amounts of 100 µL at 4 • C. According to the fluctuations of the scattered light intensity detected at a wavelength of 633 nm and a fixed angle of 173 • , the Brownian motion of each particle was measured. The peak of the Gaussian model fit to the particle distribution determined the average diameter of these exosomes and was presented by the polydispersity index (PDI) [84]. Each data point represented an average of three automatic measurements of 12-18 runs.

Exosome Uptake Using the Exo-Glow Kit
The uptake of exosomes by the cultured HUVECs was analyzed by labeling 30 µg of purified ADSC-exo using the Exo-Glow Kit (SBI Systems Bioscience, Palo Alto, CA, USA), according to the manufacturer's instructions. The Exo-Glow Kit contained exo-Red (cat. #EXOR100A-1), which is based on membrane-permeable acridine orange (AO) chemistry using fluorescently labeled single-stranded RNAs inside exosomes (typical RFP filter set), and Exo-Green (cat. #EXOG200A-1), which is based on membrane-permeable carboxyfluorescein succinimidyl diacetate ester (CFSE); activated from inside the exosomes, it is coupled to the amino ends of proteins to fluoresce green (typical GFP filter set). The uptake of exosomes was measured by incubating the labeled exosomes with 1× 10 5 cells per well in a 6-well culture plate in serum-free media for 30 min in a cell culture incubator. Subsequently, the cells were stained with 4 ,6-diamidino-2-phenylindole (DAPI mounting medium VECTOR-H1200) and detected using a confocal microscope (FLUOVIEW FV10i, Olympus).

Involved Signal Pathways Following Exosome Delivery
The effects of ADSC-LPS-exo on 45 signal transduction pathways in HUVECs were elucidated using the Cignal Finder Reporter Arrays (cat. #CCA-901 L; Qiagen). The reporter array had 45 inducible transcription factor-responsive constructs, controlling the firefly luciferase reporter gene, in duplicates, in a 96-well plate. The Renilla luciferase gene was included in the wells to normalize the transfection efficiency. To determine the effects of exosomes on the signaling pathways, the constructs were reverse-transfected into HUVECs at 8 × 10 4 cells per well in Opti-MEM ® (Invitrogen, cat. #31985-062) containing 10% FBS and 1% NEAA (Invitrogen, cat. #11140-050) using the Effectene Transfection Reagent (Qiagen, cat. #301425).

Cell Migration Assay
The migration assay was performed using two-well IBIDI TM Culture-Inserts (Ibidi, Martinsried, Germany). The silicone culture inserts were then placed in the middle of a 12-well plate. The culture plate was coated with 1% gelatin, and HUVECs transfected with 10 nM siRNA-CREB, siRNA-AP-1, siRNA-NF-κB1, or scramble siRNAs were seeded onto the culture plate at a density of 1 × 10 4 cells/mL in 70 µL volumes and allowed to proliferate to 100% confluence. The silicone insert was carefully removed 24 h later, leaving a 500 µm cell-free gap. Thereafter, ADSC-exo or ADSC-LPS-exo were added to the wells. An Olympus CKX41 microscope (Olympus) was used to capture the migration distance of cells to the cell-free zone at 0, 2, 4, 8, and 15 h. Image J was used to calculate the area of migration of cells. This study was performed with n = 6 for each condition.

Tube Formation Assay
HUVECs transfected with 10 nM siRNA-CREB, siRNA-AP-1, siRNA, NF-κB1, or scramble siRNAs in 2.5% FBS-MEM199 were seeded at a density of 1 × 10 4 cells/well in 100 µL culture medium in a 96-well plate coated with 50 µL Matrigel per well and were incubated at 37 • C in an atmosphere containing 5% CO 2 . After treatment with 30 µg ADSCexo or ADSC-LPS-exo per well, the tube formation of HUVECs at 4 and 6 h was captured in four independent experiments using an inverted phase contrast microscope (Olympus, IX71, Tokyo, Japan) at 10× magnification and quantified by Angiotool software [85] to calculate total vessel lengths and the total number of junctions. This study was performed with n = 6 for each condition.

Angiogenesis-Related Proteins
To compare the expression of angiogenesis-related proteins in HUVECs following ADSC-LPS-exo or ADSC-exo treatment, the Proteome Profiler Human Angiogenesis Antibody Array (cat. #AAH-ANG-2, RayBiotech, Dallas, TX, USA), which can detect 53 human angiogenesis-related proteins simultaneously with chemiluminescent detection reagents, was used to measure the protein expression in the cell lysates in quadruplicate, according to the manufacturer's protocol.

Extraction of Exosomal Protein and iTRAQ Labeling
Exosomal proteins of ADSC-exo and ADSC-LPS-exo were purified using the T-PER tissue protein extraction reagent (78,510, Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were desalted using Amicon ® Ultra-15 (Millipore) and quantified using the BCA protein assay (23,225, Thermo Fisher Scientific). For iTRAQ labeling, 25 µg of the protein samples were dried using SpeedVac and resuspended in the iTRAQ dissolution buffer, which included 0.5 M triethylammonium bicarbonate (TEAB; pH 8.5). Protein samples were reduced using the iTRAQ reduction buffer (tris-2-carboxyethyl phosphine, TCEP) at 60 • C for 30 min and then alkylated in the dark using iodoacetamide at 37 • C for the same amount of time. After protein digestion using sequencing-grade modified trypsin (V511A, Promega, Madison, WI, USA), the samples were dried using SpeedVac. Next, the peptides were reconstituted in the iTRAQ dissolution buffer and labeled using iTRAQ labeling reagents, according to the manufacturer's instructions (Applied Biosystems Inc., Foster City, CA, USA).

Two-Dimensional Liquid Chromatography with Tandem Mass Spectrometry (2D LC-MS/MS)
The iTRAQ-labeled samples were analyzed using a Q Exactive TM HF mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate™ 3000 RSLCnano HPLC System (Thermo Fisher Scientific). The iTRAQ-labeled peptides were pooled and desalted using Sep-Pak C18 cartridges (Waters, Milford, MA, USA). The desalted peptides were dried using SpeedVac and resuspended in 0.5% trifluoroacetic acid. The peptide mixtures were loaded onto an EASY-Spray™ C18 column (Thermo Fisher Scientific) and separated using a 0.1% formic acid solution with varying amounts of acetonitrile (5-80%). The top 15 abundant precursor ions within the 375-1400 m/z scan range were dynamically selected for further fragmentation in high collision dissociation (HCD) mode, with the normalized collision energy set to 33 ± 1%. In the full MS scan, the resolution was set to 60,000 at 200 m/z, AGC target to 3e 6 , and maximum injection time to 50 ms. For the MS/MS scan, the resolution was set to 15,000, AGC target to 5e 4 , and the maximum injection time was set to 100 ms. The release of the dynamic exclusion of selected precursor ions was set to 20 s.

Database Search and Protein Quantification
Raw MS data were examined using the Mascot search algorithm (version 2.5, Matrix Science) against the Swiss-Prot human protein database using Proteome Discoverer (version 2.1, Thermo Fisher Scientific) software. For protein identification, the search parameters were set as follows: carbamidomethylation at cysteine as the fixed modification, oxidation at methionine, acetylation at protein N-terminus, iTRAQ-labeled at peptide N-terminus, lysine residue as dynamic modifications, 10 ppm and 0.02 Da for MS/MS tolerance, and maximum missing cleavage sites with 2.

Construction of the Protein-Protein Interaction (PPI) Network and Identification of Hub Proteins
PPI network analysis [86,87] was used to distinguish critical hub proteins among groups of differentially expressed protein targets identified in the iTRAQ experiment. Therefore, the STRING database was used to conduct the PPI network analysis. PPI networks were constructed using Cytoscape 3.6.1, with nodes representing proteins and edges indicating simplifications of interactions between nodes in the network for graphical representation.

Statistical Analysis
All results are presented as the mean ± standard error. An overall analysis of the differences between group means was performed using a one-way analysis of variance (ANOVA), followed by a post hoc Fisher's least significant difference test. Statistical significance was set at p < 0.05.

Conclusions
This study revealed that human ADSCs stimulated with LPS secrete exosomes that enhance angiogenesis in HUVECs. This phenomenon is related to the activation of CREB, AP-1, and NF-κB signal transduction pathways and IL-8 production in recipient HUVECs after ADSC-LPS-exo treatment. Pre-treatment of ADSCs with LPS can produce exosomes that carry potent molecules for enhanced angiogenesis. Proteomic analysis of ADSCexo revealed the notable presence of HDAC, APP, and ITGB1, which may be potential candidates for exosome-mediated enhanced angiogenesis.